Evaluation of Relationship between Body Mass Index with Vitamin D Receptor Gene Expression and Vitamin D Levels of Follicular Fluid in Overweight Patients with Polycystic Ovary Syndrome

Background Polycystic ovary syndrome (PCOS) is the most common endocrine disorder associated with reproductive disorders and metabolic dysfunctions including insulin resistance. The roles of vitamin D in the regulation of metabolic modulations specifically involving insulin and reproduction processing are introduced. In addition, obesity appears to be closely associated with severity of PCOS. The present study is to evaluate the effect of body mass index (BMI) on vitamin D levels in follicular fluid and vitamin D receptor (VDR) expression levels in granulos cells. Materials and Methods A comparative study was conducted on 80 women with average age of 20-35 years referred for in vitro fertilization (IVF). Patients were divided into four groups, and serum levels of testosterone and insulin resistance (IR) were evaluated at the puncture time. Also, vitamin D levels of follicular fluid were evaluated. VDR gene expression was assayed by quantified-polymerase chain reaction (PCR) technique. Correlations were evaluated with calculation of the Spearman coefficient, and also independent relationships were assessed by means of multiple regression analysis. Results Vitamin D levels of follicular fluid decreased in PCOS patients compared with non-PCOS. Also, over-weight individuals had lower vitamin D levels compared with normal-weight patients. Vitamin D levels of follicular fluid were highly correlated with BMI (r=-0.51, P<0.01). Homeostatic model assessment-IR (HOMA-IR) values were significantly higher in women of PCOS/overweight and PCOS/normal weight in comparison with women of non-PCOS/normal weight (P<0.01). The gene expression data of VDR in granulosa cells were significantly lower in the PCOS/overweight group compared with the non-PCOS/normal weight (P<0.01). Conclusion The findings indicated significant differences in VDR gene expression in granulosa cells and vitamin D of follicular fluid in PCOS/overweight patients.


Introduction
Polycystic ovary syndrome (PCOS) is the most common endocrine disorder that can affect 8% to PCOS is associated not only with reproductive dismetabolic dysfunctions, including insulin resistance (5), increased oxidative stress (6), and endothelial dysfunction (7). It can be noticed that PCOS, as a heterogeneous androgen excess disorder with varying degrees of reproductive and metabolic abnormalities, is determined by the interaction of multiple genetic and environmental factors (8).
Several studies have revealed that certain metabolic disturbances such as IR and hyperinsulinemia are majour defects in the majority of PCOS patients (9, tamin D in biological processes, including regulation of cellular growth (11), differentiation, and metabolic (12,13). Among the many physiologic processes inphysiology are submitted (14). Biological actions of vitamin D are intermediated through vitamin D receptor (VDR) gene expression which is a member of the steroid/thyroid nuclear hormone receptor superfamily, displayed in calcium-regulating tissues, intestines (12), the skeleton (15), parathyroid glands (16), and reproductive tissues including ovary, uterus, pla-Additionally, investigations of animals have conits impact on the resumption and progression of follicular development. Furthermore, disturbances in calcium regulation can be responsible for follicular arrest (21). Previous study have suggested the functions of vitamin D in reproduction (22). They have also indicated that VDR regulates more than 3% of the human genome, including genes that are crucial for glucose metabolism. VDR is a transcription factor regulating the transcription of other downstream genes in many tissues that are crucial for glucose metabolism (23, 24). On the other hand, calcium are essential in the regulation of insulin secretion by b-cells. Therefore, vitamin D and VDR gene are important factors in calcium regulation and control of bcell functions, respectively. This is further supported by the fact that low vitamin D levels are associated with IR and that they can induce type-2 diabetes in PCOS patients (25). However, the exact mechanisms underlying the association of vitamin D and IR are not fully understood. One complication of PCOS is obesity which appears to be closely associated with severity of the disease phenotype (26).
In Iran, more than half of the patients with PCOS are either overweight or obese (27). It is role in the pathophysiology of hyperandrogenism, severity of insulin resistance, and also chronic anovulation (28). Increased adiposity is associated with several abnormalities of sex steroid metabolism and results in increased androgen production and suppression of sex hormone binding globulin (SHBG) (29). Thus, obesity may affect vitamin D levels in healthy women and PCOS patients. The present study aimed to evaluate the effect of obe-VDR expression levels in granulosa cells. The results of this research may contribute to the diagnosis and treatment of overweight PCOS patients.
The current comparative study was carried out on to Alzahra-Hospital of Tabriz, Iran for in vitro fertilization (IVF). Before entering the study, all the patients provided written consent forms. This study was approved by the Ethics Committee of Tabriz University of Medical Sciences (code: 5/4/2781). Weight and height of all the patients were measured, and then the body mass index (BMI, Kg/m 2 ) was calculated by dividing weight by height square. The patients were divided into two groups based the World Health Organization (WHO): the normal weight and overweight groups with the BMI of 18.5-24.9 and 25-29.9, respectively. The con-ferred for IVF due to tubal and/or male infertility or even ovulatory volunteers with normal ovaries.
IVF. These patients were recognized based on Rotterdam Consensus criteria and had at least two of the three following criteria: ovulatory disturbance, hyerandrogenism, and more than twelve 2 to 9 mm follicles in each ovary. The exclusion criteria were as follows: history of menstrual disorders such as cycle length either less than 25 days or more than 35 days, patients with other endocrine disorders or neoplastic causes of hyperandrogenemia such as androgen-secreting tumors (serum testosterone lev--For pituitary down-regulation, the patients were of gonadotropin releasing hormone (GnRH) agonist (Lucrin, Abbott Pharmaceuticals, Kurnell, Australia), depending on the age of every woman. When at least three follicles reached a diameter of almost 17 mm and the levels of peripheral plasma estradiol chorionic gonadotropin (hCG, Profasi, Serono, Aubonne, Switzerland) was given as a single IM injection. Thirty six hours after hCG administration, the oocytes were retrieved and collected with sterile Pasteur pipettes, and then the remainder of the tubes for subsequent isolation of granulosa cells.
D levels were measured in the supernatant. 25-OH vitamin D was estimated by chemiluminescence enzyme immunoassay (IDS, Boldon, UK). The blood samples were obtained before the operation for subsequent biochemical analyses. These samples were analyzed for follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, prolactin, insulin, and glucose levels.
terwards, 4 ml of phosphate buffer saline (PBS) was added to the pallet. After mixing, the solution was macia Biotech, Uppsala, Sweden). The sample was blood cells. After the centrifugation, the granulosa cells were placed between PBS and Percoll solutions. The cells were harvested by gentle pipetting, and also washed several times with PBS, and used for RNA extraction and cDNA synthesis. The total RNA of the collected samples was the recommend-ed protocol of manufacture. In brief, 1 ml of RNX plus was added to the sample in a clean RNase-free tube. The sample was homogenated via gentle up and downing and then was incubated for 5 minutes RNA was transferred to a clean RNase-free tube.
propanol was added to it; the sample was then in-let, including the total RNA, was washed using 8 minutes. After drying ethanol, the RNA pellet was water. The concentration of total RNA was calcumeans to address the purity of RNA. was electrophoresed. The genomic DNA was removed from the extracted RNA by adding RNase free DNase I (Thermo, Fermentase). The cDNA was synthesized with Moloney murine leukemia virus Aldrich Co., UK) according to the manufacture's protocol. As soon as the RNA was isolated from the granulosa cells, the reverse transcriptase reactions were performed on all the samples to generate cDNA.
To quantify the mRNA expression levels of VDR gene in the granulosa cells, real-time polymerase chain reaction (RT-PCR) was performed on a Bio-Rad iQ5 system (Bio-Rad Laboratories, Hercules, USA), using EVA-Green quantitative PCR mix kit (Sinaclon, Tehran-Iran). GAPDH gene was used as reference standard gene for all analyses to control the amount of the synthesized cDNA. PCR reactions were carried out in triplicate for each sample, and then the mean of the three readings was taken as fold-induction value. Fold change (X) values equation, in which ues of the target genes and the Ct values of the ref-standard gene. The sequence of PCR primers for amplifying VDR and GAPDH genes is provided in Table 1. IR was estimated, using the homeostatic model assessment-IR (HOMA-IR) method. In addition, HOMA-IR was calculated as the product of the fasting plasma insulin value (mU/mL) and the fasting plasma glucose value (mg/dL). Insulin levels were also estimated by ELISA kit (Siemens, Erlangen, Germany) according to the manufacture's recommendations.
All statistical procedures were run, using SPSS-16 tion of data was evaluated through the one-sample Kolmogorov-Smirnoff test. The comparisons of the means were performed by one-way ANOVA and the general linear model multi-variance by posthoc analysis for pairwise comparisons. Correlations were evaluated by calculating the Spearman coefvia multiple regression analysis. Table 1 shows the variables measured in PCOS patients. The statistical Kolmogorov-Smirnoff test proved a normal distribution for the measured parameters. The multiple regression analysis was applied to examine the relationship between obesity and testosterone, HOMA-IR levels, vitamin D lev-VDR gene expression on gramulosa cells. The results demonstrated that vitapatients and over-weight individuals compared with non-PCOS and normal-weight patients. The results also revealed that 25 OH-D levels of follicu-and PCOS/normal weight than group those in nondifference in HOMA-IR values between the women in non-PCOS/overweight and non-PCOS/normal   Table 2 represents the correlation between age, BMI, and HOMA-IR and the number of follicles, D levels, and VDR expression levels of granulosa cells. Furthermore, there was a considerable positive correlation between HOMA-IR and BMI terone levels in PCOS/overweight patients were not substantially higher than those in non-PCOS/ - Quantitative RT-PCR results exhibited a lower VDR gene expression in PCOS patients compared to the control group (Fig.1).

Fig.1:
VDR - The fold change in the expression of the target gene, VDR, was normalized to GAPDH in the PCOS/overweight, PCOS/normal weight, and non-PCOS/overweight groups. Afterwards, its expression in the control group, non-PCOS/normal weight, was analyzed. Melting curve analynot shown). A negative strong correlation was found between VDR expression levels and BMI (Table 3). The gene expression data of VDR in times) in PCOS/overweight group in comparison level of VDR expression in PCOS/overweight group was similar to that of VDR expression in PCOS/normal weight group.

Discussion
Two PCOS related complications are obesity and IR. Studies which show low vitamin D levels are associated with IR, and administration of vitamin D may ameliorate insulin sensitivity; however, the mechanisms of this effect are not clear 1-a-hydroxylase activity and serum 1,25(OH) 2D3 levels in response to parathyroid hormone (PTH), while 1,25(OH) 2D3 is observed to act like a genomic stimulator of the insulin response in the control of glucose transport (31). Therefore, vitamin D may exert a positive effect on insulin action by stimulating the expression of insulin receptor, thus stimulating insulin responsiveness for glucose transport. Additionally, vitamin D responsive element is present in the promoter of the human insulin gene, and the transcription of insulin gene is activated by 1,25(OH) 2D3 (32). In accordance with the previous study, the current results con--tive correlation between BMI increase and vitamin the overweight PCOS individuals had lower vitawith other patients. Recent reports have indicated possible mechanisms for lower serum 25-OH-D3 associated with obesity. IR and obesity are also related to a reduction in growth hormone (GH) secretion in PCOS patients. This could be accounted for decreased levels of 1,25(OH) 2D3 because expression and, consequently, serum 1,25(OH) VDR a negative correlation between vitamin D levels correlation was not substantial, its value level was imperative. Studies have also shown that obesity has been consistently associated with vitamin D study by the negative correlation of weight and The relationship between allelic variation of VDR in pancreatic island and insulin secretion and glucose tolerance indicates a role for vitamin D in the pathogenesis of IR (36). Furthermore, vitamin D administration can improve insulin sensitivity and decrease insulin level (37). Accordingly, it is logical to state that in PCOS patients with increased BMI, vitamin D levels and VDR gene expression -VDR gene expression and HOMA-IR, which is in agreement with previous reports (38).
Based on the recent research, 25(OH) D levels are correlated with androgen levels in men, and one might speculate on an association of vitamin D with androgen. The underlying mechanisms, however, remain to be explored. The current study showed a negative correlation between vitamin D levels of it is imperative. Vitamin D levels and intracellular calcium stores may regulate serum androgen levels of PCOS patients. As a result, vitamin D has a biologically plausible role in female reproduction, including the regulation of insulin secretion (39)